Structural biology has elucidated several general principles for how the ap TCR interacts with peptide- MHC ligands, but several fundamental questions, pertaining to both the recognition and activation of the TCR, remain unanswered. First, we do not yet understand the structural basis of TCR bias for MHC. Is there a recognition code underlying the loosely convergent TCR/pMHC diagonal docking orientations, or "footprints" so far seen in the different complexes ? A recent TCR/pMHC complex structure we determined suggests there may be limited sets of docking motifs that could be elucidated through structural studies of a series of related complexes. These docking topologies are likely the result of a complex interplay between junctionally-encoded TCR CDR3 interactions with peptide, and the germline- encoded Variable region interactions with the MHC helices. Therefore, we are engineering molecules for structural studies that will isolate the contributions of each of these components. Second, what is the scope of TCR cross-reactivity, and to what extent does TCR CDR conformational flexibility play a role in expanding the T cell repertoire ? Our previous studies suggest that T cell recognition is not broadly promiscuous, and yet there is a prevailing notion that CDR3 induced fit is a mechanism to expand the repertoire of pMHC recognized by the TCR. In order to better understand the role of conformational dynamics in TCR/pMHC interactions, we are determining TCR and pMHC structures, and solution interactions, using Nuclear Magnetic Resonance spectroscopy (NMR). Finally, we currently do not know how TCR and CDS interact in the TCR-CD3 complex. How is recognition of pMHC by the TCR structurally communicated to the associated CD3 subunits for subsequent signaling ? We propose to reconstitute and purify a recombinant form of a full-length, membrane-bound TCR-CD3 complex for biophysical imaging studies. In summary, during the previous term of this award we developed robust methods for expression of TCR, and peptide-MHC that now enable us to carry out a multi-disciplinary analysis of a focused set of TCR/pMHC interactions using x-ray crystallography, NMR, peptide libraries, phage display, and electron microscopy (EM).